p68 RNA helicase is a member ofthe DEAD box family proteins which share a conserved an Aspartic acid–Glutamic acid–Alanine–Aspartic acid(D-E-A-D) sequence motif (Hirling, Scheffner et al. 1989). There are many members in the DEAD family, such as p68, p72 and eIF-4a. All the members of the DEAD box family have a conserved amino acid motif, which includes the conserved DEAD box (Petry, Johnson et al. 1995; Shin, Rossow et al. 2007; Wilson and Giguere 2007). The conserved regions can be
found in many species, such as mouse, human, Escherichia coli, Drosophila and Saccharomyces cerevisiae (Jost, Schwarz et al. 1999). The DEAD box family proteins
show RNA binding and unwinding activities through the conserved DEAD box amino acids sequence (Iggo and Lane 1989). DEAD box proteins have been shown to be involved in a wide range of biological processes involving interaction with specific RNA substrates; examples include pre-mRNA splicing, ribosome biogenesis, translation and embryogenesis (Stevenson, Hamilton et al. 1998).
Many homologs of p68 RNA helicase have been discovered in chickens, yeast, frogs, human and mice. In humans, p68 RNA helicase is encoded by a singlegene locus. The transcribed partof human p68 gene is ~6.7 kb long and is split into 13 exons. Human p68 RNA helicase was first identified as an antigen cross reacting with a monoclonal antibody to SV40 large-T antigen, which is a DNA dependent ATPase (Huang and Liu 2002).
P68 RNA helicase plays important roles in pre-mRNA splicing process (Huang and Liu 2002; Liu 2002). Pre-mRNA splicing is an accurate process, which requires four small nuclear ribonucleoprotein particles (snRNPs): U1, U2, U4/U6 and U5 as well as some other non-snRNPs proteins. U1 snRNP binds to the 5’ splicing site of pre-mRNA and U2 snRNP binds to the branch point. The spliceosome is formed when the U4/U6/U5 triRNPs joins the intermediate spliceosome complex. The 5’ splice site is cleaved and the exposed 5’ end of the intron links to the branching site. Next, the 3’-OH end of the first exon adds to the 5’ end beginning site of the second exon. The whole intron between the two exons is cut off and released as a lariat and the two exons are jointed together to form a mature mRNA. p68 RNA helicase unwinds the transient U1-5’ splice site duplex during
the spliceosome assembly process. The ATPase and helicase activities of p68 are required for the dissociation of U1 snRNA from the 5’ slice site, which indicates that p68 RNA helicase might play roles in unwinding U1 snRNA and 5’ splicing site complex. p68 RNA helicase might have functions in the formation of the spliceosome with the adding of U4/U6 and U5 snRNPs, which does not require the ATPase and helicase activities of p68 (Lin, Yang et al. 2005). As an RNA helicase, p68 is required for microRNA duplex unwinding, which is required for the unwinding of the transient RNA duplex to single stranded RNA, to take into the Argonaute protein to perform its silencing activity (Salzman, Shubert-Coleman et al. 2007). p68 RNA helicase plays important roles in early organ development and maturation. In mouse embryonic fibroblasts, the lack of p68 is related to the depression of many microRNAs (Salzman, Shubert-Coleman et al. 2007).
In the nucleus, p68 RNA helicase interacts with many other proteins to regulate its gene transcription as a co-activator (Warner, Bhattacherjee et al. 2004; Fuller-Pace, Jacobs et al. 2007). p68 interacts with p53 through the C-terminal region of p53 to regulate p53 downstream targeting genes, such as p21, Fas, PIG3 and mdm2 (Bates, Nicol et al. 2005). p68 RNA helicase is reported to be recruited to pS2 gene promoter, a downstream target of estrogen receptor alpha (ERα), to regulate its transcription (Watanabe, Yanagisawa et al. 2001). In these studies, neither the ATPase activity nor the RNA helicase activity of p68 is required for its co-activator function. p68 also interacts with CBP through the C-terminal of CBP to enhance CBP-mediated transcription (Rossow and Janknecht 2003). p68 RNA helicase interacts with Smad3, specifically the MH2 domains of Smad3, to form an active transcription regulator complex (Warner,
Bhattacherjee et al. 2004). Recently it is reported that p68 RNA helicase interacts with the androgen receptor (AR) in the nucleus and regulate gene transcription as a co- activator for androgen-dependent and androgen-independent prostatic malignancy (Clark, Coulson et al. 2008).
There are many post-translational modifications of p68 RNA helicase in mammalian cells, such as sumoylation, polyubiquitylation and phosphorylation. p68 was first reported to be polyubiquitylated in colorectal cells (Causevic, Hislop et al. 2001). Sumoylation is found at a single site of p68, Lysine 53, which is enhanced by PIAS1 (protein inhibitor of activated STAT1) (Jacobs, Nicol et al. 2007). The sumoylated p68 co-represses gene transcription through interacting with HDAC1 for p300 and Elk1 (Ets- like kinase 1) (Jacobs, Nicol et al. 2007). Recombinant p68 RNA helicase purified from
E.Coli, is phosphorylated at Serine, threonine and Tyrosine residues (Yang and Liu
2004). In many cancer cells, p68 is phosphorylated at Tyrosine residues but not in their corresponding normal cell lines (Yang, Lin et al. 2005). p68 is phosphorylated at the Tyrosine 593 residue under the stimulation of PDGF-β in colorectal cells. The Tyrosine phosphorylated p68 blocks the phosphorylation of β-catenin by GSK-3β and displaces Axin from β-catenin distruction complex. The released β-catenine is shuttled into the nucleus by p68 RNA helicase which interacts with the LEF/TCF site to regulate gene expressions, which are required for EMT process (Yang, Lin et al. 2006). p68 is also double phosphorylated at Tyrosine 593 and Tyrosine 595 residues in T98G glioblastoma cells as well as HeLa cells. The double phosphorylation of p68 facilitates its apoptosis resistance to some TRAIL-induced apoptosis. The double phosphorylation of p68 RNA
helicase protects T98G cells from caspase-8 activation to trigger apoptosis (Yang, Lin et al. 2007).
Immunohistochemical examination shows p68 RNA helicase is dominantly localized in the nucleus in mammalian cells. p68 is detected in the cytosol depending on the cell cycle (Yang, Lin et al. 2007). Under the stimulation of growth factors, such as EGF, PDGF, some p68 comes out of the nucleus into the cytosol. According to the sequence analysis, there are many putative NES and NLS signals in p68. Two NES and two NLS functional signals are discovered in p68 RNA helicase sequence. Interestingly, the shuttling capability of p68 RNA helicase is not dependent on its ATPase activity. The nucleocytoplasmic shuttling function of p68 is CRM-1 dependent.